Directed Evolution of the Asparaginase Enzyme to Alter Substrate Specificity

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Yousefi, Danial | 2020

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Type of Document: M.Sc. Thesis
Language: Farsi
Document No: 52828 (03)
University: Sharif University of Technology
Department: Chemistry
Advisor(s): Kalhor, Hamid Reza
Abstract:
Asparagainase is a therapeutic enzyme which has been a subject of research for decades. The enzyme catalyzes the hydrolysis of the amide group in asparagine and similar amides. Altering the substrate specificity and stabilization of this enzyme can increase its therapeutic properties. Moreover, asparaginases may be evolved to catalyze the hydrolysis of other similar compounds. These can be achieved through directed evolution and computational methods.In this study, the gene encoding L-asparaginase II enzyme from E. coli was amplified by polymerase chain reaction (PCR) and was cloned into an expression vector. The recombinant protein was expressed by an appropriate host secreting the recombinant protein into the growth medium, and its activity was confirmed.The enzyme's active site was rationally studied for altering substrate specificity. Different approaches were used to find potentially thermostabilizing mutations. A platform was set up and optimized to evolve the enzyme for altered substrate specificity through directed evolution by using a ligation-free random mutagenesis method and a suitable selection system
Keywords:
Recombinant Protein ; Asparaginase Enzyme ; Directed Evolution ; Protein Engineering ; Protein Stabilization ; Substrate Specificity